Type I antiestrogens, e.g. tamoxifen (TAM), are used to prevent recurrence of estrogen-dependent disease in women with breast cancer and have beneficial agonist effects in other estrogen target tissues. TAM, and its metabolite 4-hydroxyTAM(4-OHT), compete with estradiol (E2) for binding to estrogen receptor (ER), activating the ER and enhancing its binding to specific DNA sites, estrogen response elements (ERE). The precise molecular mechanisms allowing ERE-containing genes to be selectively responsive to estrogens versus ligands for other nuclear receptors and bind EREs, e.g., COUP-TF and retinoic acid receptors, are not well understood. Preliminary evidence suggests that the nature of the ERE sequence acts as an allosteric effector, altering ER conformation and thus modulating liganded-ER interaction with coactivators and components of the transcription initiation complex to regulate gene expression. The proposed studies will test the following hypotheses: 1) ERE sequence and that of its immediate adjacent sequences impact ER binding and conformation and impact ligand binding. I propose that ligand binding stability is important for maintaining ER in a conformation necessary for transactivation of target gene expression. Results will correlate DNA sequence, ligand binding stability, and alterations in ER sensitivity to trypsin digestion with transcriptional activation in transiently transfected cells. 2) The distance between ERE half-sites and the nature of immediately adjacent flanking sequences regulate ER versus type II nuclear or orphan receptor binding. Preliminary studies show ER binds more avidly to ERE half-sites located on opposite faces of the DNA helix. Results will provide detailed analysis of how ERE half-site spacing contributes to estrogen and antiestrogen action. 3) ER synergizes with Sp1 to regulate the transcription of two natural estrogen target genes: creatinine kinase B and lactoferrin by direct interactions that increase ER-ERE binding affinity and stability. Results will reveal the basis for how ER ligand and the sequence of the ERE and its surrounding nucleotides, impact ER conformation, ERE binding affinity, ER interaction with Sp1, and ligand- dependent induction of gene expression in vivo.